For cultivation of cells, particularly eukaryotic cells, and more specifically mammalian cells, there is a constant need to use special cell culture media that provide nutrient substances that are required for efficient growth of the cells and for the production of the recombinant proteins or an immunogenic composition comprising a virus or virus antigen. For the efficient production of biological products, such as viruses or recombinant proteins, it is important that an optimal cell density is achieved as well as the protein expression itself is increased to obtain maximum product yield.
Cell culture media formulations have been supplemented with a range of additives in the past, including undefined components like fetal calf serum (FCS), several animal derived proteins and/or protein hydrolysates of bovine origin.
In general, serum or serum-derived substances, such as albumin, transferrin or insulin, may contain unwanted agents that can contaminate the cell cultures and the biological products obtained there from. Furthermore, human serum derived additives have to be tested for all known viruses, including hepatitis and HIV, that can be transmitted by serum. Moreover, bovine serum and products derived there from bear the risk of BSE contamination. In addition, all serum-derived products can be contaminated by unknown constituents. In the case of serum or protein additives that are derived from human or other animal sources in cell culture, there are numerous problems (e.g. the varying quality in composition of the different batches and the risk of contamination with mycoplasma, viruses or BSE), particularly if the cells are used for production of drugs or vaccines for human administration.
Therefore, many attempts have been made to provide efficient host systems and cultivation conditions, which do not require serum or other animal protein compounds. Simple serum free medium typically includes basal medium, vitamins, amino acids organic or inorganic salts, and optionally additional components to make the medium nutritionally complex.
Plant or yeast hydrolysates are known to be useful for fermentation processes and can enhance the growth of many fastidious organisms, yeasts and fungi. WO 96/26266 describes that gluten hydrolysate can be used in tissue culture. Franek et al. (Biotechnology Progress (2000) 16, 688-692) describe growth and productivity promoting effects of defined soy hydrolysate peptide fractions.
WO 96/15231 discloses a serum-free medium composed of the synthetic minimal essential medium and yeast extract for propagation of vertebrate cells and virus production process. A medium formulation composed of a basal cell culture medium comprising a rice peptide and an extract of yeast and enzymatic digest thereof, and/or a plant lipid for growth of animal cells is disclosed in WO 98/15614. WO 00/03000 discloses a medium that comprises a soy hydrolysate and a yeast extract, but also requires the presence of recombinant forms of animal proteins, such as growth factors.
WO 01/23527 discloses a medium for the animal protein-free and serum-free cultivation of cells, especially mammalian cells, whereby the medium contains soy hydrolysate.
However, since plant and yeast hydrolysates are natural products, the quality of commercially available lots of these plant or yeast hydrolysates varies extremely. The quality differs dependent on the growth, maturity and harvest of the starting plants or yeast. Of course, for plant hydrolysates also those features are dependent from the area of cultivation, in particular climate-induced variations. Further, the different suppliers in the market use different manufacturing processes and also the products of a single supplier vary from lot-to-lot.
In large scale cell culture one can observe that, even if, there seems to be no difference in the quality of different commercially available lots of those hydrolysates by regular means of quality control, like e.g. the given specification of the producer, there is a considerable difference in the performance of the hydrolysate with respect to cell growth, protein expression rates and virus propagation, respectively, when these hydrolysates are used as supplements of basal cell culture media. As a result, there are large variations in the production of recombinant proteins or viral products as a function of the quality of lots of plant or yeast hydrolysates, in particular soy hydrolysates used (“lot-to-lot variation”) as a supplement of basal cell culture media or as a component of a cell culture medium.
To solve this problem, WO 2006/045438 discloses a “plant or yeast hydrolysate reduced” medium in order to obtain more consistent characteristics in the respective media due to lower plant or yeast hydrolysate contents. Such media comprise a reduced amount of plant or yeast hydrolysate in combination with polyamines,
Nevertheless, even in those “plant or yeast hydrolysate reduced” media the performance differences based on the lot-to-lot variation of those hydrolysates are still considerable. In this context the term “lot” means a production batch provided by a specific manufacturer, wherein the mass and volume, respectively, can vary from manufacturer to manufacturer. In case of soy hydrolysate a lot is several tons.
In order to determine the performance of a plant or yeast hydrolysate in advance, the respective lots of said hydrolysate are used in a small scale cell culture process/test (“use test”) prior to the use in large scale process. Only lots having a high performance characteristic in the small scale cell culture tests are used later on in large scale processes.
Such small scale cell culture tests are very time consuming and cost intensive due to the need of laboratory resources. For instance, about six weeks are needed to perform the cell culture tests and analysis thereof, involving about twenty staff members in laboratory. Due to this time consuming small scale cell culture tests for performance evaluation of the plant or yeast hydrolysate, it is not possible to purchase only high performing lots from the supplier. After a six week evaluation time, normally, the supplier has already sold all lots under investigation. For a large scale biotechnological campaign involving, e.g. a 6000 liter fermenter more than one lot is required. Therefore, normally a few lots have to be purchased and stored while performing the small scale cell culture tests. The low performing lots are not used in large scale processes because of the risk of failure. However, the costs for purchasing these lots have already been arisen. In addition, in most cases the low performing lots have to be disposed of which also raises significant costs and wastes natural resources.
Even if, small scale cell culture tests will also be necessary in the future in order to exclude the presence of inhibitory components in those plant or yeast hydrolysates etc., there is a need for a fast pre-screening method for performance prediction of a given plant or yeast hydrolysate lot to avoid or at least significantly reduce the number of small scale cell culture processes/tests in order to save time and resources.
In order to analyze lot-to-lot variability in raw materials like protein or yeast hydrolysates José et al. (Biotechnol. Prog., 2011, Vol. 27, No.5, pages 1339 to 1346) combined near and mid infrared spectroscopy with two-dimensional (2D)-fluorescence spectroscopy. For that reason an aqueous solution of a specific peptone as raw material was analyzed in comparison to a defined aqueous chemical media containing multiple components. As a result it was found that 2D-fluorescence spectroscopy alone was only suitable to analyze and/or predict the performance of a defined chemical media. Moreover, the 2D-fluorescence spectra of the tested raw materials could only be weakly correlated with the actual measured performance in cell culture media. The authors concluded that only a combination of these methods can be used for performance prediction.
Non-published experiments showed that 2D-fluorescence spectroscopy measurements of aqueous solutions of plant or yeast hydrolysate samples showed no significant differences in different samples which could be correlated to certain production performance characteristics, manufacturing parameters or productivity parameters.
Surprisingly, it was found that when the 2D-fluorescence spectroscopy measurements of powder samples of the respective plant or yeast hydrolysates were taken, the resulting spectra showed great signal strength as well as broad variation of signals. What is even more beneficial is that no time consuming sample preparation is needed, e.g. preparing an aqueous solution of the respective plant or yeast hydrolysate followed by sterilization. Further, the amount of plant or yeast hydrolysate material used for the method according to the invention can be immensely reduced by a factor of about 20 to about 50 in comparison to the prior art measurements using aqueous solutions of the respective plant or yeast hydrolysate samples. Therefore, e.g. a small amount of soy hydrolysate powder from a lot can be used as a sample to predict the performance of the whole lot.
Furthermore, these 2D-fluorescence spectroscopy results of a given plant or yeast hydrolysate sample, in particular derived from a plant or yeast hydrolysate lot, could be correlated with manufacturing data of a protein when produced in a cell culture medium comprising an amount of said plant or yeast hydrolysate lot.
A benefit of the invention is also the fact that there is no need to combine various spectral analysis methods as described in the prior art.